Water soluble taxol derivatives

ABSTRACT

Alkaline sensitive protaxol is water soluble and is hydrolyzed at physiological (alkaline) pH to render the native taxol structure and the native taxol activity. Protaxol compositions include 2&#39;- and/or 7-O-ester derivatives of taxol and/or 2&#39;- and/or 7-O-carbonate derivatives taxol. Protaxol has a formula as follows: ##STR1## wherein R 1  and R 2  are each H or a radical selected from the group consisting of --CO--(CH 2 ) m  --X--(CH 2 ) n  --COZ and --COO--(CH 2 ) o  --Y--Ar, and wherein m, n, and o are each an integer of 1 to 3; X is O, S, NH, SO, or SO 2  ; Y is S, SO or SO 2  ; Ar is phenyl or substituted phenyl wherein the substituent is halo, amino, nitro or N,N-dialkylamino having 1 to 4 carbons in each of the alkyl groups; and Z is OH, OR 3 , SR 3  or NR 4  R 5  wherein R 3  is alkyl containing 1 to 4 carbons and R 4  and R 5  are each alkyl containing 1 to 4 carbons, or taken together with the nitrogen to which they are attached form a saturated heterocyclic ring having 4 or 5 carbons, with the proviso that at least one R 1  and R 2  is not hydrogen; as well as the salts of such compounds with organic/inorganic bases and acids, preferably pharmaceutically acceptable salts.

This is a divisional, of application Ser. No. 08/122,722, filed Sep. 16,1993, now U.S. Pat. No. 5,422,364 which is a continuation-in-part ofU.S. application Ser. No. 07/940,535, filed Sep. 4, 1992 now abandoned,and of PCT/U.S. 93/08397, filed Sep. 7, 1993, whose disclosures areincorporated herein by reference.

SPECIFICATION

1. Field of Invention

The invention relates to water soluble derivative of taxol which arehydrolyzable at alkaline pH and which, upon hydrolysis, possessantitumor activity against a wide variety of carcinoma cells. Moreparticularly, this invention relates to protaxol compositions including2'- and/or 7-O-ester derivatives and/or 2'- and/or 7-O-carbonatederivatives of taxol, pharmaceutical compositions comprising the sameand methods of preparing the same.

This work was assisted through the financial support of the NationalInstitutes of Health (CA 46446). Accordingly, the U.S. Government hascertain rights to this invention.

2. Background of the Invention

Taxol (1) is a natural product isolated from the Pacific yew tree (Taxusbrevifolia). It was first isolated in 1971 from the western yew, Taxusbrevifolia by Wani et al. (J. Am. Chem. Soc., 1971, 93, 2325), whocharacterized its structure by chemical and X-ray crystallographicmethods. Taxol was recently approved for treatment of ovarian cancerpatients. Insolubility problems with this drug, however, have promptedattempts to improve its pharmacological profile.

Taxol and various taxane derivatives (collectively herein referred to as"taxols") are highly cytotoxic and possess strong in vivo activities ina number of leukemic and tumor systems. Especially, taxol (1) isconsidered an exceptionally promising cancer chemotherapeutic agent, andis currently in phase II clinical trials in the United States. Equallyimportant is taxotere (2) (L. Mangatal et al., Tetrahedron, 1989, 45,4177), a semisynthetic analog of taxol which is also undergoing clinicaltrails with impressive results. Clinical results have demonstrated highefficacy of taxols against such cancer types as ovarian, lung, gastric,breast, colon and cervical carcinomas. Taxol is a member of the taxanefamily of diterpenes having the following structure: ##STR2##

A synthetic analog of taxol has the following structure: ##STR3##

Taxol is only slightly soluble in water and this has created significantproblems in developing suitable pharmaceutical formulations useful forchemotherapy. Some formulations of taxol for injection or I.V. infusionhave been developed utilizing CREMOPHOR EL® (polyoxyethylated castoroil) as the drug carrier because of taxol's aqueous insolubility. Forexample, taxol supplied by the NCI has been formulated in 50% CREMOPHOREL® and 50% dehydrated alcohol. CREMOPHOR EL®, however, is itself toxicand produces, when given in a large volume single dose without taxol,vasodilation, labored breathing, lethargy, hypotension and death indogs. Therefore, the use of this carrier would not be recommended.

In an attempt to increase taxol's solubility and to develop more safeclinical formulations, studies have been directed to synthesizing taxolanalogs where 2'- and/or 7-position is derivatized with groups thatwould enhance water solubility. These efforts yielded protaxol compoundsthat are more water soluble than the parent compound and that deplay thecytotoxic properties upon activation.

U.S. Pat. No. 4,942,184 to R. D. Haugwitz, et al. discloses watersoluble taxols having variously substituted acyl groups at2'-O-position.

U.S. Pat. No. 4,960,790 to V. J. Stella, et al. discloses water solubletaxols the 2'- and/or 7-hydroxyl of which is derivatized with a selectedamino acid or an amino acid mimetic compound.

Structure-activity relationships and solubility improvements of taxolhave been probed through substitution at the C-2' hydroxyl group. Forexample, H. M. Deutsch et al. disclosed the synthesis of water-solubleprodrugs of Taxol with potent antitumor activity, Journal of MedicalChemistry (1989) 32, 788-792. More recent efforts to this end were alsodisclosed by Charles Swindell et al. (Journal of Medical Chemistry(1991) 34, 1176-1184), by Zhiyang Zhao et al. (Journal of NaturalProducts (1991) 54, 1607-1611) and by Abraham E. Mathew et al. (Journalof Medical Chemistry (1992) 35, 145-151). However, none of the prodrugsdisclosed in the prior art exhibited sensitivity to alkaline hydrolysisunder physiological conditions. This absence of sensitivity compromisestheir utility as pharmacological agents.

Accordingly, it is apparent that it would have been desirable to developprotaxol derivatives which would be more water soluble than taxol, butwhich, upon hydrolysis under physiological (alkaline) conditions, wouldexhibit the same or similar level of antitumor activity as unmodifiedtaxol. Furthermore, the rate of hydrolysis should facilitate thepharmacokinetics of the drug so as to enhance its delivery. The presentinvention achieves this goal by providing protaxol derivatives that arewater soluble and that are susceptible, under physiological conditions,to a novel mechanism of controlled hydrolysis for providing thebioactive form of taxol at a pharmacokinetically favorable rate.

SUMMARY OF THE INVENTION

A series of taxol-releasing compounds (protaxols) with improvedproperties are synthesized and demonstrated to have enhanced biologicalactivity. These prodrugs are designed on the basis of chemicalprinciples to allow for in vitro and in vivo taxol release under basicor physiological conditions. Chemical studies demonstrate the stabilityof these compounds at pH greater than 7 and the ability of thesecompounds to release taxol in basic media. Biological investigationsconfirm the taxol-like activity of these prodrugs as microtubulestabilizing agents. Studies with tumor cell lines reveal cytotoxicproperties comparable to those of taxol, whereas incubation with humanblood plasma confirm rapid taxol release. These results demonstrate thepotential of such designed compounds as anticancer agents with improvedprofiles as compared to those of taxol and Taxotere (2).

Organic chemistry concepts are employed as guiding principles torationally designed two types of structures (FIG. 1B). Recognizing thatthe C-2' hydroxyl group is the most convenient site to attach designedfunctional domains and realizing that blocking this position wouldresult in loss of activity, groups fulfilling the following criteria areselected:

(a) The functional group must initiate its own cleavage from theconjugate and must generate taxol in situ upon suitable activation; and

(b) The newly introduced group must increase the water solubility of thecompound.

Since certain drug-resistant tumor cells exhibit basic pHmicroenvironments, base labile moieties are preferred as activatinggroups. Both types of appendages illustrated in structures I and II arecleaved either by basic conditions or via enzymatic assistance asindicated in FIG. 1B.

Protaxol compounds belonging to the Type I group include a water solublefunctional group having a carbonate ester attached to the C-2' positionof taxol. The water soluble functional group is connected to the C-2'position by means of a carbonate ester. Also connected to the carbonateester is an electron withdrawing aryl substituent. Under physiologicalconditions, the rate of taxol activation of Type I compounds increaseswith the electron withdrawing ability of the aryl substituent. Asillustrated in FIG. 1B, hydrolysis may be induced by physiological oralkali conditions and proceeds with an elimination reaction resulting inthe release from the carbonate ester of both the aryl substituent andthe taxol. Activation of protaxols from Type I compouds necessarilyresults in the evolution of CO₂.

Protaxol compounds belonging to Type II group include a water solublefunctional group attached by means of an ester linkage to the C-2'position of taxol. The distal end of the water soluble functional groupincludes a carboxylic group. Between the carboxylic group at the distalend and the ester linkage adjacent to the taxol is an electronwithdrawing heteroatom or group. Under physiological (alkaline)conditions, the water soluble functional group undergoes a cyclizationinvolving the formation of an anhydride or anhydride intermediate. Therate of hydrolysis and taxol activation increases with the electronwithdrawing nature of the linking heteroatom.

It is, therefore, an object of the present invention to provide taxolderivatives (protaxol) with enhanced water solubility while retainingthe ability to reconstitute or reactivate the parent taxol and itscytotoxic properties.

It is another object of the present invention to provide methods ofpreparing water soluble derivatives of taxol with antitumor activity.

It is a further object of the present invention to provide a method oftreating tumors in a mammal by administration of water solublederivatives of taxol.

It is another object of the present invention to provide methods forconverting protaxol to taxol.

It is a still further object of the present invention to provide apharmaceutical composition comprising an effective antitumor amount ofwater soluble derivatives of taxol as an active ingredient and apharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates the molecular structure of taxol and Taxotere (TM).

FIG. 1B illustrates the mechanistic rationale for the design ofprotaxols.

FIG. 2A illustrates the kinetics of taxol release from protaxol 5 inaqueous buffer solutions at various pH's and temperatures.

FIG. 2B illustrates the kinetics of taxol release from protaxol 5 inhuman blood plasma and pH 7.4 buffer solution at 37° Centigrade.

FIGS. 3A to 3D illustrate the tubulin polymerization-depolymerizationmeasurements with taxol and designed protaxol 5.

DETAILED DESCRIPTION OF THE INVENTION

The taxol derivatives 3-12 illustrated in Table 1A were designed andsynthesized on the basis of the above considerations, The carbonateswere prepared via derivatization with an appropriate (2-thioaryl)ethylchloroformate while the esters were formed by reaction with a suitableanhydride. Further manipulations yielded additional compounds fortesting.

The solubilities of the synthesized protaxols in D₂ O were determined by1H NMR spectroscopy. These studies revealed considerably highersolubilities (Table 1A) than taxol for compounds of type II whilecompounds of type I (FIG. 1B) showed little improvement in watersolubility as compared to taxol.

All designed compounds exhibited excellent stability in aprotic organicsolvents and in the solid state. However, in aqueous media, some ofthese compounds showed tendencies to slowly revert to taxol even at pH7.0. The derivatives in which the C-7 hydroxyl group was acetylated wereextremely unstable in aqueous media losing the C-7 substituent veryrapidly. The stability of the synthesized compounds was examined atvarious pH's and temperatures. FIG. 2A summarizes such a study carriedout with protaxol 5 in which the accelerating effect of highertemperatures and pH on taxol release was demonstrated. Table 1A includesthe half-lives of six of these compounds at pH 7.5 and 9. As seen fromthese data, for compounds of type I, the rate of taxol release increaseswith the electron withdrawing ability of the aryl substituents, whereasfor type II derivatives, the rate of release increases with the electronwithdrawing nature of the linking heteroatom. This illustrates theability to fine-tune the rate of release of taxol to a desired set ofconditions using simple chemical principles.

                                      TABLE 1                                     __________________________________________________________________________    Designed protaxols and their solubility and stabilty properties (A) and       cytotoxicity data for selected compounds (B).                                  ##STR4##                                     A                                                              Water solubility                                                                      1.sub. 1/2 n                                                                       1.sub. 1/2 n                      Compound                       (mg/mL).sup.k                                                                         (pH 7.5)                                                                           (pH 9)                            __________________________________________________________________________    1: taxol: R.sup.1 = R.sup.2 = H                                                                              <<0.10                                          ##STR5##                 ; R.sup.2 = H                                                                      0.84    >500 110                                ##STR6##                 ; R.sup.2 = H                                                                      0.35    >500 300                                ##STR7##                 ; R.sup.2 = H                                                                      1.2.sup.m                                                                             >500  30                                ##STR8##                 ; R.sup.2 = H                                                                      --      --   --                                 ##STR9##                 ; R.sup.2 = H                                                                      --      --   --                                 ##STR10##                ; R.sup.2 = H                                                                      <0.10.sup.1                                                                           >500  66                                ##STR11##                ; R.sup.2 = H                                                                      <0.10.sup.1                                                                           >500  7                                 ##STR12##                ; R.sup.2 = H                                                                      <0.10.sup.1                                                                           >>500                                                                              101                                ##STR13##                --   0.50    --   --                                 ##STR14##                --   0.46    --   --                                __________________________________________________________________________

                  TABLE 1B                                                        ______________________________________                                        Cytotoxicity of taxol derivatives (IC.sub.50 [M])*                            ______________________________________                                        Against Normal Cell Lines                                                                Compounds:                                                         Normal Cell lines                                                                          Taxol(1) 5        6      7                                       ______________________________________                                        NHDF         10.sup.-6                                                                              10.sup.-6                                                                              10.sup.-6                                                                            10.sup.-6                               Normal human skin                                                             RPMI-7666    10.sup.-4                                                                              10.sup.-4                                                                              10.sup.-4                                                                            10.sup.-4                               Normal human PBLs                                                             BALB/c 3T3   10.sup.-6                                                                              10.sup.-5                                                                              10.sup.-5                                                                            10.sup.-5                               Normal mouse                                                                  embryo                                                                        CHO          10.sup.-4                                                                              10.sup.-4                                                                              10.sup.-4                                                                            10.sup.-4                               Chinese hamster                                                               ovary                                                                         CHO          10.sup.-4                                                                              10.sup.-4                                                                              10.sup.-4                                                                            10.sup.-4                               Normal human                                                                  mammary                                                                       ______________________________________                                                   Compounds:                                                         Normal Cell lines                                                                          9        10       11     12                                      ______________________________________                                        NHDF         10.sup.-6                                                                              10.sup.-6                                                                              10.sup.-6                                                                            10.sup.-6                               Normal human skin                                                             RPMI-7666    10.sup.-4                                                                              10.sup.-4                                                                              10.sup.-4                                                                            10.sup.-4                               Normal human PBLs                                                             BALB/c 3T3   10.sup.-5                                                                              10.sup.-5                                                                              10.sup.-6                                                                            10.sup.-5                               Normal mouse                                                                  embryo                                                                        CHO          10.sup.-4                                                                              10.sup.-4                                                                              10.sup.-4                                                                            10.sup.-4                               Chinese hamster                                                               ovary                                                                         CHO          10.sup.-4                                                                              10.sup.-4                                                                              10.sup.-4                                                                            10.sup.-4                               Normal human                                                                  mammary                                                                       ______________________________________                                        Against Cancer Cell Lines                                                                Compounds:                                                         Cancer cell lines                                                                          Taxol(1) 5        6      7                                       ______________________________________                                        SK           10.sup.-5                                                                              10.sup.-5                                                                              10.sup.-5                                                                            10.sup.-5                               Melanoma                                                                      Capan-1      <10.sup.-9                                                                             <10.sup.-9                                                                             10.sup.-7                                                                            <10.sup.-9                              Pancreatic                                                                    H322         10.sup.-9                                                                              10.sup.-9                                                                              10.sup.-7                                                                            10.sup.-9                               Lung carcinoma                                                                MCF-7        <10.sup.-5                                                                             10.sup.-5                                                                              10.sup.-5                                                                            10.sup.-5                               Breast carcinoma                                                              BT-549       10.sup.-9                                                                              <10.sup.-9                                                                             10.sup.-9                                                                            <10.sup.-9                              Breast carcinoma                                                              OVCAR-3      <10.sup.-9                                                                             <10.sup.-9                                                                             10.sup.-6                                                                            <10.sup.-9                              Ovarian carcinoma                                                             HT-29        <10.sup.-9                                                                             <10.sup.-9                                                                             10.sup.-7                                                                            <10.sup.-9                              Colon carcinoma                                                               SIHA         10.sup.-5                                                                              10.sup.-4                                                                              10.sup.-4                                                                            10.sup.-5                               Cervix carcinoma                                                              786-0        10.sup.-7                                                                              10.sup.-6                                                                              10.sup.-5                                                                            10.sup.-5                               Renal cell carcinoma                                                          PC-3         10.sup.-6                                                                              10.sup.-6                                                                              10.sup.-6                                                                            10.sup.-6                               Prostate carcinoma                                                            HL-60        <10.sup.-9                                                                             <10.sup.-9                                                                             10.sup.-7                                                                            <10.sup.-9                              Promyel carcinoma                                                             MOLT-4       <10.sup.-9                                                                             10.sup.-9                                                                              10.sup.-7                                                                            <10.sup.-9                              T-cell leukaemia                                                              L1210        10.sup.-6                                                                              10.sup.-5                                                                              10.sup.-5                                                                            10.sup.-5                               Mouse leukaemia                                                               UCLA-P-3     <10.sup.-9                                                                             <10.sup.-9                                                                             <10.sup.-9                                                                           <10.sup.-9                              Lung carcinoma                                                                ______________________________________                                                   Compounds:                                                         Cancer cell lines                                                                          9        10       11     12                                      ______________________________________                                        SK           10.sup.-5                                                                              10.sup.-5                                                                              10.sup.-5                                                                            10.sup.-5                               Melanoma                                                                      Capan-1      10.sup.-6                                                                              <10.sup.-9                                                                             <10.sup.-9                                                                           <10.sup.-9                              Pancreatic                                                                    H322         10.sup.-6                                                                              10.sup.-8                                                                              10.sup.-9                                                                            10.sup.-7                               Lung carcinoma                                                                MCF-7        10.sup.-5                                                                              10.sup.-5                                                                              10.sup.-5                                                                            10.sup.-5                               Breast carcinoma                                                              BT-549       10.sup.-7                                                                              10.sup.-7                                                                              <10.sup.-9                                                                           <10.sup.-9                              Breast carcinoma                                                              OVCAR-3      10.sup.-6                                                                              <10.sup.-9                                                                             10.sup.-6                                                                            10.sup.-8                               Ovarian carcinoma                                                             HT-29        10.sup.-7                                                                              <10.sup.-9                                                                             <10.sup.-9                                                                           10.sup.-7                               Colon carcinoma                                                               SIHA         10.sup.-5                                                                              10.sup.-5                                                                              10.sup.-5                                                                            10.sup.-5                               Cervix carcinoma                                                              786-0        10.sup.-6                                                                              10.sup.-6                                                                              10.sup.-6                                                                            10.sup.-6                               Renal cell carcinoma                                                          PC-3         10.sup.-6                                                                              10.sup.-6                                                                              10.sup.-6                                                                            10.sup.-6                               Prostate carcinoma                                                            HL-60        10.sup.-7                                                                              <10.sup.-9                                                                             <10.sup.-9                                                                           <10.sup.-9                              Promyel carcinoma                                                             MOLT-4       10.sup.-9                                                                              <10.sup.-9                                                                             <10.sup.-9                                                                           <10.sup.-9                              T-cell leukaemia                                                              L1210        10.sup.-6                                                                              10.sup.-5                                                                              10.sup.-6                                                                            10.sup.-6                               Mouse leukaemia                                                               UCLA-P-3     <10.sup.-9                                                                             <10.sup.-9                                                                             10.sup.-9                                                                            10.sup.-9                               Lung carcinoma                                                                ______________________________________                                    

Significantly, incubation of protaxol 5 in human blood plasma at 37°Centigrade demonstrates accelerated release of taxol, with a half-life(t.sub. 1/2) of approximately 100 minutes, as compared to the slowerrate of taxol release in aqueous media under the same conditions of pHand temperature (FIG. 2B). This finding supports the chemical assistancefor taxol release from this protaxol 5 by factors present in human bloodplasma and raises expectations for in vivo efficacy of this compound.

Tubulin polymerization to microtubules is promoted by GTP, whereas CaCl2causes depolymerization of microtubules back to tubulin (FIG. 3A). Taxolallows and promotes this type of microtubule assembly and, furthermore,it stabilizes microtubules against CaCl2-induced depolymerization (FIG.3B). The designed protaxol 5 was tested for its ability to stabilizemicrotubules formed from tubulin via the action of GTP. This agentfailed to prevent CaCl2-induced disassembly of microtubules at theinitial stages of the experiment (FIG. 3C), but showed increased potencywith prolonged exposure times as expected from the slow release oftaxol, and reached potencies comparable to that of taxol whenessentially complete conversion to taxol had occurred (FIG. 3D). Similarresults were obtained with a number of other designed taxols shown inTable 1. These findings are in agreement with the previously reportedloss of activity resulting from blocking the C-2' hydroxyl group.

The protaxols synthesized in this study were tested against a broadspectrum of cell lines ranging from the multiple-drug resistant ovarian(OVCAR-3) to lung (H-322) and leukemia (MOLT-4) cells in order to assesscytotoxicity, cell-type selectivity, and duration of action. Comparisonswith taxol itself revealed not only similar potencies but also similarselectivities against various cell lines. This is consistent with amechanism of action involving taxol release rather than the derivativeitself exhibiting intrinsic activity. Furthermore, taxol was isolatedand characterized from aqueous solutions of the designed compounds, thusfirmly establishing the release of the agent under the conditions of themicrotubule assembly and cytotoxicity experiments. The most significantfindings are shown in Table 1B. As demonstrated by these results, therather impressive selectivity of taxol against cancer cells versusnormal cells is maintained in the designed compounds. Noteworthy are thelower cytotoxicities of compounds 6 and 9 after a three-day exposurewhich are in line with the lower rates of release of taxol from theseconjugates.

The results presented herein demonstrate the power of chemicalprinciples in the molecular design of fine-tuned derivatives of thetaxol family for biological and pharmacological studies. The designedmolecules act as prodrugs, releasing the active agent in situ as shownby chemical studies. Investigations with tubulin and in vitrocytotoxicity experiments confirmed the unique taxol-like profiles of anumber of these compounds. Particularly promising is protaxal 5 whichexhibited good solubility and stability properties in aqueous mediawhile rapidly releasing taxol in human blood plasma. Issues remaining tobe addressed include further pharmacological studies with thesecompounds and the design and synthesis of more potent and selectivecompounds as well as effective agents against resistant-type tumors.Such agents may be arrived at by modifications of naturally occurringcompounds or by total synthesis.

Accordingly, the present invention is directed to novel taxolderivatives having the general formula (I): ##STR15## wherein R¹ and R²are each H or a radical selected from the group consisting of--CO--(CH₂)_(m) --X--(CH₂)_(n) --COZ and --COO--(CH₂)_(o) --Y--Ar,wherein m, n, and o are each an integer of 1 to 3; X is O, S, NH, SO, orSO₂ ; Y is S, SO or SO₂ ; Ar is phenyl or substituted phenyl wherein thesubstituent is halo, amino, nitro or N.N-dialkylamino having 1 to 4carbons in each of the alkyl groups; and Z is OH, OR³, SR³ or NR⁴ R⁵wherein R³ is alkyl containing 1 to 4 carbons and R⁴ and R⁵ are eachalkyl containing 1 to 4 carbons, or taken together with the nitrogen towhich they are attached form a saturated heterocyclic ring having 4 or 5carbons, with the proviso that at least one of R¹ and R² is nothydrogen; as well as the salts of such compounds with organic/inorganicbases and acids, preferably pharmaceutically acceptable salts.

The expression "saturated heterocyclic ring having 4 or 5 carbons" usedherein refers to groups such as 1-pyrrolidinyl, 1-piperidinyl,1-morpholino, 1-thiomorpholino.

The present invention includes (a) derivatives esterified at the2'-hydroxyl group of taxol, (b) derivatives esterified at the 7-hydroxyl group of taxol, and (c) derivatives esterified at both the2'-and 7-position hydroxyl groups.

In the above formula (I) preferred values for R¹ and R² are --CO--CH₂--X--CH₂ --COOH wherein X is as previously defined. Other preferredvalues are --COO--(CH₂)₂ --Y--C₆ H₅ wherein Y is as previously defined.

Thus, a first preferred group of compounds of the present invention arethose wherein R² is H and R¹ is --CO--CH₂ --X--CH₂ --COOH wherein X isas previously defined.

A second preferred group of compounds of the present invention are thosewherein each of R¹ and R² is --CO--CH₂ --X--CH₂ --COOH wherein X is aspreviously defined.

A third preferred group of compounds of the present invention are thosewherein R² is hydrogen and R¹ is --COO--(CH₂)₂ --Y--C₆ H₅ wherein Y isas previously defined.

These novel derivatives of formula (I) may be prepared by esterifyingtaxol with an appropriate acid HO--CO--(CH₂)_(m) --X--(CH₂)_(n) --COZ ora haloformate of Hal--COO--(CH₂)_(o) --Y--Ar wherein X, Y, Z, and Ar areas previously defined and Hal is halo. The acid is normally activatedprior to reaction with taxol, involving formation of a symmetricanhydride, mixed anhydride, activated ester, acid chloride or the like.Particularly preferred acid reagents used in the present invention arediglycolic anhydride and thiodiglycolic anhydride. The activation stepcan be carried out by various condensing agents, such ascarbonyldiimidazole, dicyclohexylcarbodiimide, hydroxybenzotriazole andthe like. Alternatively, the esterification between taxol and the acidcan be conducted in the presence of such condensing agents.

The esterification may be facilitated by addition of a catalyst such aspyridine, 4-N,N-dimethylaminopyridine (DMAP) or triethylamine althoughthe catalyst is not always required.

Thus, the esterification is conducted in a reaction-inert solvent in thepresence of a condensing agent (if no prior activation of an acid isinvolved) with or without the additional presence of a catalyst at atemperature of from 0° to about 100° C., preferably at ambienttemperature.

Suitable reaction-inert solvents which can be used in the esterificationare aprotic solvents such as N,N-dimethylformamide,N,N-dimethylacetamide, N-methylpyrolidone, and hexamethyl phosphoramide;aromatic solvents such as benzene, toluene, xylene, ethylbenzene, andchlorobenzene; chlorinated hydrocarbons, such as chloroform anddichloromethane; ethers, such as diethyl ether and tetrahydrofuran; lowmolecular weight esters, such as ethyl acetate and butyl acetate; lowmolecular weight aliphatic ketones, such as acetone and methyl ethylketone, and mixture thereof. It is, however, especially advantageous touse pyridine as the solvent, since it also catalyzes the esterificationstep.

In practice taxol is usually treated with an excess of the acid or thehaloformate, preferably 3 to 10 moles of each per mole of taxol. Thereaction time varies according to a number of factors, but at about 25°C. reaction times from 0.5 to 24 hours are commonly used. The compoundsof formula (I) can be isolated and purified by conventional methodsknown in the art, e.g., chromatography.

Since the 2'-hydroxyl of taxol is more reactive than the 7-positionhydroxyl, the 2'-position is esterified first. Thus, the aboveesterification leads to the compounds of formula (I) wherein R¹ is otherthan hydrogen, i.e. 2'-O-ester derivatives. When more than 2 equivalentsof the acid or the haloformate are used in the reaction, theesterification may provide 2'-, 7-disubstituted taxol derivatives.Although the esterification at the 2'-position can be conducted with orwithout a catalyst, the esterification at the 7-position may require thepresence of a catalyst due to the reduced chemical reactivity of the7-hydroxyl of taxol.

For the preparation of 7-O-esters of taxol, the 2'-hydroxyl must beprotected or blocked, then the 7-hydroxyl is esterified and further the2'-protecting or blocking group is removed. A wide variety of hydroxylprotecting groups can be employed for this purpose. Exemplary of suchprotecting groups are trialkylsilyl where each alkyl contains 1 to 5carbons, methoxymethyl, 1-ethoxyethyl, benzyloxymethyl,tetrahydropyranyl, and 2,2,2-trichloroethoxy-carbonyl.

The reaction between 2'-protected taxol and the esterifying agentproceeds in essentially the same manner as described above. Theprotecting groups can then be removed by appropriate means known in theart (e.g. mild acid, mild base, treatment with fluorides orhydrogenolysis).

The starting acids HO--CO--(CH₂)_(m) --X--(CH₂)_(n) --COZ and thehaloformate Hal--COO--(CH₂)_(o) --Y--Ar are either known compounds ormay be prepared by methods reported in the prior art.

In the case of compounds of formula (I) wherein Z is other thanhydroxyl, they can conveniently be prepared by reacting acids of formula(I) wherein Z is OH with an appropriate amine (H₂ NR⁴ R⁵), alcohol, (R³OH) or thiol (R³ SH).

Compounds of formula (I) wherein X and Y are each SO or SO₂ can beobtained from compounds of formula (I) wherein X and Y are each S or SOby means of oxidation. Oxidants which can be employed in this oxidationinclude peracids such as peracetic acid, perbenzoic acid,m-chloroperbenzoic acid, and monoperphthalic acid, hydrogen peroxide,periodic acids such as paraperiodic acid, and sodium periodate, andinorganic oxidants such as potassium monopersulfate.

The oxidation can be carried out in a reaction inert solvent at atemperature of from 0° to about 50° C. Suitable solvents includedichloromethane, chlorform and lower alcohols (methanol or ethanol).When potassium monopersulfate is used as the oxidant, the oxidationreaction may be carried out in an aqueous medium utilizing a watermiscible solvent such as methanol. The reaction times are in the rangeof about 0.5 to 24 hours. For one mole of the reactant, it is preferredto use at least one molar equivalent of the oxidant. For the preparationof the sulfone, the use of an excess amount of the oxidant is desirable.The sulfones can be obtained simply by adding excess oxidant to asolution of the crude sulfoxide formed and allowing oxidation tocompletion. Alternatively, the sulfoxides can be prepared directly fromthe thio compounds of formula (I) wherein X or Y is S using excessoxidant.

The product sulfoxide or sulfone can be isolated and purified byconventional methods known in the art, e.g., chromatography.

The oxidation may produce a mixture of sulfone and sulfoxide. By properchoice of molar ratios and reaction conditions, the reaction may bestopped at the sulfoxide stage. Thus, the selective preparation ofsulfoxides of formula (I) wherein X or Y is SO is possible. Also, adisastereomic mixture of the sulfoxide of formula (I) wherein X or Y isSo will result from the above oxidation reaction. If desired, thediasteromers can be separated by column chromatography, since theydiffer markedly in polarity. These diastereomers are to be consideredwithin the scope and purview of the present invention.

Compounds of formula (I) wherein Z is OH, have a carboxyl group andtherefore they will form pharmaceutically acceptable salts withinorganic and organic bases. These base salts are prepared by standardmethods, for example by contacting the acidic and basic components in astoichiometric ratio, in an aqueous, non-aqueous or partially aqueousmedium, as appropriate. They are then recovered by filtration, byprecipitation with a non-solvent followed by filtration, by evaporationof the solvent, or, in the case of aqueous solutions, by lyophilization,as appropriate. Illustrative are the ammonia salt, alkali salts, andorganic amine salts which are preferred. Examples of the organic aminesalts include trimethylamine, triethylamine, triethanolamine,N-methyl-N,N-diethanolamine, N-methylglucamine.

Compounds of formula (I) having an amino or N,N-dialkylamino group mayform pharmaceutically acceptable salts with inorganic and organic acids.Of particular value are the sulfate, hydrochloride, hydrobromide,nitrate, phosphate, citrate, tartrate, pamoate, perchlorate,sulfosalicylate, benzenesulfonate, 4-toluenesulfonate, and2-naphthalenesulfonate salts.

The taxol derivatives of formula (I) can be utilized in the treatment oftumors in a mammal due to their cytotoxic, antitumor activity. Thesecompounds can be administered to a mammal, particularly human by eitherthe oral or parenteral routes of administration.

Where gastrointestinal absorption permits, oral administration ispreferred for reasons of patient convenience and comfort. In general,these taxol derivatives are normally administered in dosages rangingfrom about 0.1 mg to about 10 mg per kg of body weight per day;variations will necessarily occur depending upon the condition of thesubject being treated and the particular compound being administered.Typically, treatment is commenced at a low daily dosage and increased bythe physician only if necessary. It is to be noted that these compoundsmay be administered in combination with pharmaceutically acceptablecarriers by either of the routes previously indicated, and that suchadministration can be carried out in both single and multiple dosages.

For parenteral use, the present compounds are formulated according tothe known art using suitable dispersing or wetting agents and suspendingagents. The sterile injectable formulation can also be a solution ofsuspension in a nontoxic parenterally acceptable diluent or solvent, forexample, as a solution in 1,3-butandiol. Among the acceptable vehiclesand solvents are water, Ringer's solution and isotonic NaCl solution,fixed oils including synthetic mono-or di-glycerides, fatty acids suchas oleic acids, and mixtures thereof.

For oral administration, a wide variety of dosage forms are used, e.g.,tablets, capsules, lozenges, trochees, hard candies, powders, syrups,aqueous suspension, elixirs, syrups, and the like formulated withvarious pharmaceutically-acceptable inert carriers. Such carriersinclude solid diluents or fillers, sterile aqueous media and variousnon-toxic organic solvents, etc. In general, the compounds of thepresent invention are present in such oral dosage forms at concentrationlevels ranging from about 0.5% to about 90% by weight of the totalcomposition, in amounts which are sufficient to provide the desired unitdosage. Tablets may contain various excipients such as sodium citrate,calcium carbonate and calcium phosphate, along with variousdisintegrants such as starch (preferably potato or tapioca starch),alginic acid and certain complex silicates, together with binding agentssuch as polyvinylpyrrolidione, sucrose, gelatin and acacia.Additionally, lubricating agents such as magnesium stearate, sodiumlauryl sulfate and talc are often very useful for tabletting purposes.Solid compositions of a similar type may also be employed as filters insoft and hard-filled gelatin capsules; preferred materials in thisconnection would also include lactose or milk sugar as well as highmolecular weight polyethylene glycols. When aqueous suspensions and/orelixirs are desired for oral administration, the essential activeingredient therein may be combined with various sweetening or flavoringagents, coloring matter or dyes and, if so desired, emulsifying and/orsuspending agents, together with such diluents as water, ethanol,propylene glycol, glycerin and various like combinations thereof.

The present compounds can also be administered in the form of liposomes.As is known in the art, liposomes are generally derived fromphospholipids or other lipid substances. Liposomes are formed by mono-ormulti-lamellar hydrated liquid crystals that are dispersed in an aqueousmedium. Any non-toxic, pharmaceutically acceptable and metabolizablelipid capable of forming liposomes can be used. The present compositionsin liposome form can contain stabilizers, preservatives, excipients, andthe like in addition to the agent. The preferred lipids are thephospholipids and the phosphatidyl cholines (lecithins), both naturaland synthetic.

Methods of forming liposomes are known in the art. See, for example,Methods in Cell Biology; Prescott, Ed; Academic: New York, 1976, Vol.XIV.

Additional pharmaceutical methods may be employed to control theduration of pharmacological action. Controlled release preparations maybe achieved by the use of polymers to complex or adsorb the presentactive compounds. The controlled delivery may be exercised by selectingappropriate macromolecules (for example, polyester, polyamino acids,polyvinyl pyrrolidone, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, and protamine sulfate) and the concentration ofmacromolecules as well as the methods of incorporation in order tocontrol release.

Another possible method to control the duration of action by controlledrelease preparations is to incorporate the present compound intoparticles of a polymeric material such as polyesters, polyamino acids,hydrogels, poly (lactic acid) or ethylene vinylacetate copolymers.Alternatively, instead of incorporating the active compounds into thesepolymeric particles, it is possible to entrap the active compounds inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly (methylmethacrylate) microcapsules,respectively, or in colloidal drug delivery systems, for example,albumin microspheres, microemulsions, nanoparticles, and nanocapsules orin macroemulsions. Such teachings are disclosed in Remington'sPharmaceutical Science, A. Oslo, Ed; 17th ed.; Mack: Easton, Pa., 1985.

After administration to a mammal by either the oral and parenteralroute, a compound of formula (I) quickly breaks down in vivo atphysiological pH to liberate taxol. Thus, the compounds of formula (I),because of their good antitumor activity and water solubility, will finduse as a prodrug of taxol.

The present invention is illustrated by the following examples. However,it should be understood that the invention is not limited to the detailsof these examples. Proton nuclear magnetic resonance spectra (NMR) weremeasured at 500 MHz unless otherwise indicated for solutions indeuterochloroform (CDCI₃) and peak positions are expressed in parts permillion (ppm) downfield from tetramethylsilane. The peak shapes aredenoted as follows: s, singlet; d, doublet; t, triplet; q, quartet; m,multiplet; and b, broad.

EXAMPLE 1 ##STR16##

A solution of taxol (2 mg, 2.36 μmol) in pyridine (0.2 ml) was treatedwith diglycolic anhydride (5 mg, 42 μmol) and was stirred for 2 hours at25° C. Then pyridine was azeotroped with benzene (3×), the residue wasstirred with water for 20 minutes and extracted with CHCl₃. The organicphase was washed with brine, dried over Na₂ SO₄ and the solvent wasremoved in vacuo. The residue was recrystallized from CHCL₃ /benzene togive the title compound as a white solid; mp 150°-152° C.

1H NMR: δ 1.14 (s, 3H, CH₃), 1.25 (s, 3H, CH₃), 1.68 (s, 3H, CH₃), 1.88(m, 1H, CH₂), 1.94 (s, 3H, CH₃), 1.97 (s, 1H, OH), 2.23 (m, 1H, CH),2.24 (s, 3H, CH₃), 2.40 (dd, J=9, 15 Hz, 1H, CH₂), 2.45 (s, 1H, OH),2.48 (s, 3H, CH₃), 2.51 (ddd, J=7, 7, 15 Hz, 1H, CH₂), 3.82 (d, J=7 H,1H, CH), 4.07 (d, J=16.5 Hz, 1H, OCH₂), 4.14 (d, J=16.5 Hz, 1H, OCH₂),4.21 (d, J=8.5 Hz, 1H, OCH₂), 4.23 (d, J=16.5 Hz, 1H, OCH₂), 4.29 (d,J=16.5 Hz, 1H, OCH₂), 4.32 (d, J=8 Hz, 1H, OCH₂), 4.44 (dd, J=7, 11 Hz,1H, OCH), 4.98 (dd, J=2, 10 Hz, 1H, OCH), 5.62 (d, J=3 Hz, 1H, OCH),5.69 (d, J=7 Hz, 1H, OCH), 6.07 (dd, J=2, 9.5 Hz, 1H, CHN), 6.28 (t, J=9Hz, 1H, OCH), 6.30 (s, 1H, OCH), 7.11 (d, J=9.5 Hz, NH), 7.45 (m, 10H,aromatic), 7.61 (tt, J=1.5, 7.5 Hz, 1H, aromatic), 7.74 (dd, J=1.5, 7Hz, 2H, aromatic), 8.14 (dd, J=1.5, 7 Hz, 2H, aromatic).

HRMS for C₅₁ H₅₅ NO₁₈ Cs (M+Cs⁺): calcd 1102.2473, found 1102.2440.

EXAMPLE 2 ##STR17##

A solution of taxol (2 mg, 2.36 μmol) in pyridine (0.2 ml ) was treatedwith thiodiglycolic anhydride (8 mg, 60 μmol ) and a catalytic amount ofDMAP. After stirring at 25° C. for 12 hours pyridine was azeotroped withbenzene (3x), the residue was stirred with water for 20 minutes andextracted with CHCL₃. The organic phase was washed with brine, driedover Na₂ SO₄ and the solvent was removed in vacuo. The residue waspurified by preparative TLC (10% MeOH in CH₂ Cl₂, Rf=0.35) to give thetitle compound (1.6 mg, 63%) as a white solid.

¹ H NMR: δ 1.11 (s, 3H, CH₃), 1.21 (s, 3H, CH₃), 1.68 (s, 3H, CH₃), 1.88(m, 1H, CH₂), 1.94 (s, 3H, CH₃), 2.10 (m, 1H, CH₂), 2.23 (s, 3H, CH₃),2.37 (m, 1H, CH₂), 2.48 (s, 3H, CH₃), 2.55 (m, 1H, CH₂), 3.18 (d, J=15Hz, 1H, SCH₂), 3.31 (d, J=14.5 Hz, 1H, SCH₂), 3.37 (d, J=14.5 Hz, 1 H,SCH₂), 3.49 (d, J=15 Hz, 1H, SCH₂), 3.80 (d, J=7 Hz, 1H, CH), 4.21 (d,J=8 Hz, 1H, OCH₂), 4.2 (d, J=8 Hz, 1H, OCH₂), 4.44 (dd, J=7, 11 Hz, 1H,OCH), 4.99 (d, J=7.5 Hz, 1H, OCH), 5.48 (d, J=3.5 Hz, 1H, OCH), 5.69 (d,J=7 Hz, 1H, OCH), 6.04 (dd, J=3.5, 9 Hz, 1H, CHN), 6.25 (t, J=9 Hz, 1H,OCH), 6.29 (s, 1H, OCH), 7.42 (m, 10 H, aromatic), 7.61 (m, 1H,aromatic), 7.82 (dd, J=1, 8.5 Hz, 2 H, aromatic), 8.15 (d, J=7 Hz, 2 H,aromatic), 8.62 (s, br, 1H, OH).

HRMS for C₅₁ H₅₄ NO₁₇ S (M-H⁺): calcd 984.3112; found 984,3123.

EXAMPLE 3 ##STR18##

A solution of 2'-thiodiglycolic taxol (EXAMPLE 2) (30.5 mg. 31 μmol) inMeOH (2 ml) was added to a solution of OXONE® (Aldrich Chemicals,Milwaukee, Wis.; 37 mg, 60 μmol) in phosphate buffer (pH=5) at 0° C. Thesolution was warmed to ambient temperature and stirred for 12 hours.Then MeOH was evaporated, the water phase extracted with CHCL₃ (3×),washed with brine, dried over Ha₂ SO₄ and the solvent was removed invacuo. After having subjected to preparative TLC (SiO₂, 16% MeOH in CH₂CL₂), the title sulfoxide derivative 3a and the sulfone derivative 3bwere isolated as beige powders. 3a:Rf=0.24 (16% MeOH in CH₂ CL₂), (4.2mg, 14%).

HRMS for C₅₁ H₅₄ NO₁₈ SCs (M-H⁺, 2 Cs⁺): calcd 1266.1170, found 1266.1130.

3b:Rf=0.36 (16% MeOH in CH₂ CL₂), (15.4 mg, 50%).

EXAMPLE 4 ##STR19##

To a solution of 2-phenylsulfone-ethanol (560 mg, 3 mmol) andtriphosgene (297 mg, 1 mmol) in CH₂ CI₂ was added 1 eq. of pyridine (243μl, 3 mmol) at 0° C. After stirring for 5 minutes the solution waswarmed to ambient temperature and stirred for another 45 min. Three eq.of this solution (88 μl, 58.5 μmol) were added to a solution of taxol(10 mg. 11.7 μmol) in CH₂ CL₂. At 0° C., pyridine (5 μl 58 μmol) wasadded. The solution was stirred at 25° C. for 12 hours, then washed withdiluent. HCL (pH=3) and brine. The organic phase was dried over Na₂ SO₄,the solvent removed in vacuo and the residue purified by preparative TLC(5% MeOH in CH₂ CL₂, Rf=0.48) to give the title compound as a whitesolid (3 mg. 25%).

¹ H NMR: δ 1.14 (s, 3H, CH₃), 1.25 (s, 3H, CH₃), 1.68 (s, 3H, CH₃), 1.88(m. 1H, CH₂), 1.91 (s, 3H, CH₃), 2.22 (m, 1H, CH₂), 2.23 (s, 3H, CH₃),2.39 (dd, J=9.5, 15 Hz, 1H, CH₂), 2.43 (s, 3H, CH₃), 2.48 (s br, 1H,OH), 2.55 (m, 1H, CH₂), 3.47 (t, J=6.5 Hz, 2H, OCH₂), 3.81 (d, J=7 Hz,1H, OCH), 4.20 (d, J=8.5 Hz, 1H, OCH₂), 4.32 (d, J=8.4 H₂, 1H, OCH₂),4.44 (dd, J=7, 11 Hz, 1H, OCH), 4.48 (t, J=6.5 Hz, 2H, SCH₂), 4.97 (d,J=7.5 Hz, 1H, OCH), 5.38 (d, J=3 Hz, 1H, OCH), 5.69 (d, J=7 Hz, 1H,OCH), 5.97 (dd, J=3, 9.5 Hz, 1H, CHN), 6.28 (t, J=9 Hz, 1H, OCH), 6.29(s, 1H, OCH), 6.82 (d, J=9.5 Hz, 1H, NH).

EXAMPLE 5 ##STR20##

A solution of taxol (4 mg. 4.7 μmol) in 0.2 ml dry pyridine was treatedwith thiodiglycolic anhydride (6.6 mg, 50 μmol) and DMAP (1.2 mg, 10μmol). After stirring at 25° C. for 12 hours, the solvent was evaporatedand the residue was stirred in water for 20 minutes. The precipitationwas filtered, dissolved in CHCL₃, washed with brine and dried over Na₂SO₄. The solvent was removed in vacuo and the residue was purified bypreparative TLC (30% MeOH in CH₂ CL₂) to give the title compound as awhite solid (3.3 mg, 64%).

¹ H NMR: δ 1.14 (s, 3H, CH₃). 1.26 (s, 3H, CH₃), 1.80 (s, 3H, CH₃), 1.88(m, 1H, CH₂), 1.94 (9s, 3 H, CH₃), 2.14 (s, 3H, CH₃), 2.22 (m, 2H, CH₂),2.39 (s, 3H, CH₃), 2.59 (m, 1H, CH₂), 3.41 (m, 8H, SCH₂), 3.87 (d, J=7Hz, 1H, CH), 4.15 (d, J=8.5 Hz, 1H, CH₂), 4.30 (d, J=8.5 Hz, CH₂), 4.95(d, J=9 Hz, 1H, CH), 5.62 (m, 3H, CH), 5.99 (dd, J=5, 9 Hz, 1H, CH),6.15 (m, 1H, CH). 6.17 (s, 1H, CH). 7.45 (m, 10H, aromatic), 7.62 (t,J=7.5 Hz, 1H, aromatic), 7.81 (dd, J=1.5, 8 Hz, 2 H, aromatic), 8.01 (d,J=7.5 Hz, 2H, aromatic), 8.64 (br s, 2H, OH).

HRMS for C₅₅ H₅₈ NO₂₀ S₂ (M-H⁺): calcd 1116.2994, found 1116.3-061.

EXAMPLE 6 Tablet Formulation

The following ingredients are combined in the following proportions byweight:

    ______________________________________                                        taxols               20                                                       lactose              200                                                      hydroxypropylmethylcellulose                                                                       5                                                        sodium starch glycolate                                                                            20                                                       magnesium stearate   5                                                        ______________________________________                                    

The mixture is blended to a uniform powder and compressed into tabletsin measured volumes corresponding to 250 mg by weight to yield tabletsof desired potency.

EXAMPLE 7 Capsule Formulation

The following ingredients are combined in the following proportions byweight:

    ______________________________________                                        taxols            50                                                          cornstarch        447                                                         magnesium stearate                                                                              3                                                           ______________________________________                                    

The mixture is thoroughly blended so as to obtain a uniform powder. Theresulting mix (500 mg fill weight) is filled into hard gelatin capsulesof a suitable size so as to obtain capsules of desired potency.

EXAMPLE 8 Cytotoxicity

The taxol derivatives of the present invention are particularly usefulfor the treatment of the same tumors for which taxol has been shownactive, including lung tumor, melanoma, leukemia, colon cancer andbreast cancer.

The ability of the compounds to inhibit tumors has been tested by invitro studies using various cancer cell lines.

Tumor or normal cells were cultured in 96-well plates with the testcompound in 200 μl of RPMI supplemented with 10% fetal bovine serum(Hyclone, Salt Lake City, Utah) at 37° C. in a humidified atmospherecontaining 5% CO₂ in air for 72 hours. Sulforhodamine B dissolved in 1%acetic acid was added, and the plates were incubated for 30 minutes atambient temperature, washed with 1% acetic acid, and blotted. To theair-dried plates were added 100 μl of 10 μM unbuffered TRIS base (pH10.5) with shaking. The optical densities of the plates were measuredwith a microplate reader (Molecular Devices Thermomax) at 540 nm. Theconcentration of compound required to reduce the absorbance to 50% ofcontrol samples was recorded as IC₅₀.

Results of the tests performed on several selected derivatives of taxolusing established normal or cancer cell lines are summarized in thetable.

    ______________________________________                                        CYTOTOXICITY OF TAXOL DERIVATIVES (IC.sub.50 [M])                                              Ex-     Ex-   Ex-   Ex-   Ex-                                                 ample   ample ample ample ample                              Cell Line                                                                              Taxol   1       2     3a    3b    4                                  ______________________________________                                        NHDF     2.08e-4 1.03e-4 5e-5  5e-5  1.9e-7                                                                              5e-5                               HLF-1    6.3e-5  5.95e-5 3.1e-6                                                                              2.5e-5                                                                              1.3e-5                                                                              6.3e-6                             W1-38    6.06e-5 5.19e-5 1.3e-5                                                                              2.5e-5                                                                              1.3e-5                                                                              5e-5                               CHO      5.35e-5 1.01e-4 5e-5  1e-4  1e-4  1e-4                               SK-MEL-28                                                                              1e-4    1e-4    1e-4  1e-4  1e-4  1e-4                               CAPAN-1  9.8e-8  9.8e-8  1.9e-7                                                                              8.7e-7                                                                              1.9e-7                                                                              9.8e-8                             H322     1.22e-4 2.05e-4 3.1e-6                                                                              8.3e-6                                                                              5e-6  6.3e-6                             MCF-7    6.44e-5 3.57e-4 1e-4  3.92e-5                                                                             6.1e-5                                                                              7e-5                               BT-549   4.46e-5 5.14e-5 1.6e-5                                                                              9.8e-8                                                                              9.8e-8                                                                              1e-9                               OVCAR-3  9.8e-8  9.8e-8  3.9e-7                                                                              9.8e-8                                                                              9.8e-8                                                                              9.8e-8                             HT-29    9.8e-8  1e-8    1.9e-7                                                                              9.8e-8                                                                              9.8e-8                                                                              9.8e-8                             SlHA     1.11e-3 1.12e-3 5e-5  2.5e-5                                                                              5e-5  3.1e-6                             786-0    1.84e-7 1.53e-7 6.3e-6                                                                              1.5e-7                                                                              1.3e-7                                                                              2.5e-6                             PC-3     6.91e-5 6.51e-5 1e-4  5e-5  5e-5  5e-5                               HL-60    1e-14   1e-14   1e-14 1e-14 1e-14 1e-14                              MOLT-4   1e-14   1e-14   1e-14 1e-14 1e-14 5e-12                              ______________________________________                                    

NHDF: Normal human dermal fibroblast (available from Clouelics Corp.,San Diego, Calif.); HLF-1: Normal human diploid lung; W1-38; Normalhuman diploid lung; CHO; Chinese hamster ovary; SK-MEL-28: Melanoma;CAPAN-1: Pancreatic carcinoma; H322: lung carcinoma; MCF-7; Breastcarcinoma; BT-549; Human breast ductal carcinoma; OVCAR-3: Human ovariancarcinoma; HT-29: Human adenocarcinoma, colon; SIHA: Human squamouscarcinoma crvx; 786-0: Human perirenal cell adenocarci; PC-3: Humanprostate adenocarcinoma; HL-60: Human promyelomic leukemia; MOLT-4:Human T-cell leukemia. All cell lines except NHDF are available from theAmerican Tissue Culture Collection, Rockville, Md.

Solubility

Some representative taxol derivatives of the present invention showimproved water solubility as compared to taxol which is essentiallyinsoluble in water. Where the compounds of formula (I) wherein Z is OHare not water soluble, their base salts can be prepared as hereinbeforeindicated and they can form normal aqueous solutions up to about 1%concentration.

Where the compounds of formula (I) which contain an amino orN,N-dialkylamino group are not water soluble, their acid salts can beprepared as hereinbefore indicated and they can form normal aqueoussolutions up to about 1% concentration.

The above test results indicate that the taxol derivatives of thepresent invention exhibit excellent antitumor activity. These compounds,therefore, are useful antitumor agents due to their biological activityand their increased water solubility as compared to taxol.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade without departing from the spirit or scope of the invention.

EXAMPLE 9 Synthesis of the Compounds of Table 1 A

Preferred reagents and conditions for the synthesis of compounds 3-12 ofTable 1 A from taxol (1) are indicated as follows:

(a) Compound 3: 1.1 equivalents of diglycolic anhydride in pyridine at25° C. for 2 hours (yield 75%);

(b) Compound 4: 2.0 equivalents of thiodiglycolic anhydride in pyridineat 25° C. for 4 hours (67%);

(c) Protaxol 5: same as (b), then 3.0 equivalents of Oxone, CH₃ OH/H₂ O,25° C., 12 hours (65% overall);

(d) Compound 6: same as (b), then CH₂ N₂, 3.0 equivalents of Oxone, CH₃OH/H₂ O, 25° C., 2 hours (32% overall);

(e) Compound 7: 1.2 equivalents of (2-thiophenyl)ethyl chloroformate,(C₂ H₅)₃ N,CH₂ Cl₂, 25° C., 2 hours (94%);

(f) Compound 8: same as (e), then 3.0 equivalents Oxone, CH₃ OH/H₂ O,25° C. (75% overall);

(g) Compound 9: 1.2 equivalents (2-p-nitrothiophenyl) ethylchloroformate, (C₂ H₅)₃ N,CH₂ Cl₂, 25° C., then Oxone, (74% overall);

(h) Compound 10: same as (g), then Oxone, followed by sodium dithionite,C₂ H₅ OH,H₂ O (62% overall);

(i) Compound 11: 10.0 equivalents diglycolic anhydride pyridine (87%);

(j) Compound 12: 10.0 equivalents thiodiglycolic anhydride,4-dimethylaminopyridine (as catalyst), CH₂ Cl₂ (29%).

Solubilities

(k) Water solubility of each of the indicated compounds was determinedby combining 2.0 mg sample with 1.0 ml D₂ O and sonicating for 15 min.The resulting mixture was centrifuged for 20 minutes and the supernatantsaturated solution was analysed by ¹ H NMR spectroscopy using a BrukerAMX-400 instrument and methanol or pyridine as internal standard. Theconcentration of the compound was determined by comparing severalsignals with the CH₃ signal of methanol or the NCH signal of pyridine.

(l) The detection limit for the determination of the solubilities ofcompounds 8, 9, and 10, as determined by diluting samples of protaxol 5,was found to be 0.10 mg ml⁻¹.

(m) The relatively high concentrations found for protaxol 5 during thesolubility measurements are due to the formation of hydrogels,presumably caused by the formation of micelles or related aggregates.

Half Life

The half-life (t_(1/2)) of the protaxol is defined as the time inminutes at which 50% taxol release was observed at 37° C.

Toxicities Reported in Table 1 B

The cytotoxicities were determined using the sulphorhodamine B assaymethod (Skehan, P. et al. J. Nat. Canc. Inst. 82, 1107 (1990)).

IC₅₀ [M] is defined as the molarity at which 50% of tumour cellviability was observed after 72 hours of exposure under standard tissueculture conditions.

EXAMPLE 10 Tubulin Polymerization-Depolymerization Experiments

Tubulin polymerization-depolymerization experiments with taxol anddesigned protaxol 5 are illustrated in FIG. 3. CaCl₂ -promoteddepolymerization of microtubules was suppressed by taxol and protaxol 5(after taxol release).

Methods

These measurements may be conducted at 37° C. following a recentlydeveloped procedure described by R. Merlock and W. Wrasidlo (AnalyticalBiochemistry, 1993). In each case 1.0 mM GTP may be employed to promoteinitial polymerization of tubulin.

A: control: Tubulin (1.0 mg/mL) alone; CaCl2 (0.25 mM) added after 16minutes caused depolymerization of microtubules.

B: Tubulin (1.0 mg/mL) with taxol (10-6M); CaCl2 (0.25 mM) added after16 minutes did not cause depolymerization.

C: Tubulin (1.0 mg/mL) with protaxol 5 (10-6M); CaCl2 (0.25 mM) addedafter 16 minutes caused depolymerization of microtubules.

D: Tubulin (1.0 mg/mL) with protaxol 5 (10-6M) after release of taxol(as determined by HPLC, essentially quantitative conversion).

Addition of the CaCl₂ solution is designated by the symbol (a).

EXAMPLE 11 Kinetics of Taxol Release from Protaxol 5

Kinetics of taxol release from protaxol 5 in aqueous buffer solutions atvarious pH's and temperatures is illustrated in FIG. 2 A. Kinetics oftaxol release from protaxol 5 in human blood plasma and pH 7.4 buffersolution at 37° C. is illustrated in FIG. 2B. Although it is quitestable at 20° C. and in pH 7.0 buffer solution, protaxol 5 is convertedto taxol in human plasma at 37° C. with a half-life of ˜100 min.

Conditions in FIG. 2A

▴pH 7.0, 20° C.; O, pH 7.0, 37° C.; , pH 7.5, 37° C.; □, pH 8.0, 37°C.; ▪, pH 9.0, 37° C.

Methods for FIG. 2A

Protaxol 5 was dissolved in dimethylsulphoxide (DMSO) at 25° C. andimmediately added to the appropriate phosphate buffer solution at thespecified temperature. Aliquots were analysed using a Waters Maximasystem HPLC instrument equipped with auto-injector (3.9×300 mm C₁₈column equipped with a precolumn; flow rate, 1.5 ml min⁻¹ ; eluant,gradient A-B, where A is 80% 100 mM ammonium acetate, buffer pH 6.0, andB is 100% methanol; ultraviolet detector). The per cent taxol releasedwas determined from the relative areas of the peaks corresponding toprotaxol 5 and taxol (the conversion was clean, and it was assumed thatthe two compounds have equal molar extinction coefficients).

Method for FIG. 2 B

Protaxol 5 and N-cyclohexylbenzamide (as internal standard) weredissolved in acetonitrile and immediatley added to human plasma orphosphate buffer at pH 7.4. Samples of protaxol 5 were incubated in eachmedium for specified times and then extracted with ethyl acetate asdescribed by S. M. Longnecker, et al., Cancer Treat. Rep. 71, 53 (1987).Solvent was removed and the residue dissolved in acetonitrile andanalysed by HPLC as already described. The peak areas for protaxol 5 andtaxol were normalized to that of the internal standard using acalibration curve.

Conditions in FIG. 2B

▴per cent protaxol 5 remaining in pH 7,4 phosphate buffer; , per centtaxol released in pH 7.4 phosphate buffer; Δ, per cent protaxol 5remaining in human plasma; O, per cent taxol released in human plasma.

We claim:
 1. A method for converting a protaxol to taxol wherein theprotaxol is represented by the following structure: ##STR21## where: R₁is selected from a group consisting of H and an ester radicalrepresented by --CO--(CH₂)_(m) --X--(CH₂)_(n) --COZ, wherein:m and n areeach an integer of 1 to 3; X is selected from a group consisting of O,S, NH, SO, and SO₂ ; and Z is selected from a group consisting of OH,OR³, SR³ and NR⁴ R⁵ whereinR³ is alkyl containing 1 to 4 carbons and R⁴and R⁵ are each alkyl containing 1 to 4 carbons, or taken together withthe nitrogen to which they are attached form a saturated heterocyclicring having 4 or 5 carbons; and R₂ is a radical selected from the samegroup from which R₁ is selected, with the proviso that at least one R₁and R₂ is not hydrogen; the method comprising the followingstep:contacting the protaxol with physiological conditions for inducingalkaline hydrolysis of at least one ester radical selected from thegroup consisting of R₁ and R₂ for producing taxol.
 2. A method forconverting a protaxol to taxol as described in claim 1 wherein:R¹ is anester radical selected from the group represented by the followingstructures: ##STR22## and R² is a radical selected from the groupconsisting of H and R¹.
 3. A method for converting a protaxol to taxolwherein the protaxol is represented by the following structure:##STR23## where: R₁ is selected from the group consisting of --H and anester radical represented by --COO--(CH₂)_(o) --Y--Ar, wherein:o is aninteger of 1 to 3; Y is selected from a group consisting of S, SO andSO₂ ; Ar is selected from a group consisting of phenyl and substitutedphenyl, wherein the substituted phenyl is selected from a groupconsisting of phenyls having substituents selected from a groupconsisting of halo, amino, nitro and N,N-dialkylamino substituents, theN,N-dialkylamino substituents having 1 to 4 carbons in each alkyl group;and R₂ is a radical selected from the same group from which R₁ isselected, with the proviso that at least one R₁ and R₂ is not hydrogen;the method comprising the following step:contacting the protaxol withphysiological conditions for inducing alkaline hydrolysis of at leastone ester radical selected from the group consisting of R₁ and R₂ forproducing taxol.
 4. A method for converting a protaxol to taxol asdescribed in claim 3 wherein:R¹ is an ester radical selected from thegroup represented by the following structures: ##STR24## and R² is aradical selected from the group consisting of H and R¹.
 5. A method forconverting a protaxol to taxol wherein the protaxol is represented bythe following structure: ##STR25## where: R₁ is a radical selected fromthe group consisting of--H, --CO--(CH₂)_(m) --X--(CH₂)_(n) --COZ, and--COO--(CH₂)_(o) --Y--Ar, wherein: m, n, and o are each an integer of 1to 3; X is selected from a group consisting of O, S, NH, SO, and SO₂ ; Yis selected from a group consisting of S, SO and SO₂ ; Ar is selectedfrom a group consisting of phenyl and substituted phenyl, wherein thesubstituted phenyl is selected from a group consisting of phenyls havingsubstituents selected from a group consisting of halo, amino, nitro andN,N-dialkylamino substituents, the N,N-dialkylamino substituents having1 to 4 carbons in each alkyl group; and Z is selected from a groupconsisting of OH, OR³, SR³ and NR⁴ R⁵ whereinR³ is alkyl containing 1 to4 carbons and R⁴ and R⁵ are each alkyl containing 1 to 4 carbons, ortaken together with the nitrogen to which they are attached form asaturated heterocyclic ring having 4 or 5 carbons, with the proviso thatat least one R₁ and R₂ is not hydrogen; and R₂ is a radical selectedfrom the group consisting of H and R₁, the method comprising thefollowing step:contacting the protaxol with physiological conditions forinducing alkaline hydrolysis of at least one ester radical selected fromthe group consisting of R₁ and R₂ for producing taxol.